Metagenomic Analysis of Tuberculosis in a Mummy
Little is known about the genotypes of ancient microbes, leaving many unanswered questions about how many pathogens have evolved into their modern strains. Fortunately, a recently unearthed crypt in Vac, Hungary has presented a rare opportunity to study the genome of an ancient strain of Mycobacterium tuberculosis. The sample was taken from the mummified remains of 28 year old Terrezia Hausmann, who died of tuberculosis on December 25, 1797. Although a chest X-ray did not reveal diagnostic tubercules, the wasted, cachetic appearance of the body indicated tuberculosis (malnutrition was not likely as the crypt was kept for wealthy families). The well-controlled conditions in the crypt as well as M. tuberculosis ''ability to persevere in harsh environments have kept the DNA is remarkably good condition. Nonetheless, extreme care was taken to protect the purity of the samples: clean protective clothing and gloves were worn, sterile tubes and pipettes were used in combination with molecular biology grade reagents in a lab that had never studied ''Mycoplasma ''DNA before. Metagenomic Analysis Rather than use PCR to amplify the mummified ''M. tuberculosis, ''which runs the risk of losing an amount of this rare sample with a poor primer run, a metagenomic, open-frame sequencing approach was taken using an Illumina MiSeq sequencer. 5.5 million paired-end reads were obtained. Less than 1% of these aligned with human DNA and 8% aligned with the ''M. tuberculosis ''H37Rv reference strain, the researchers called this "deep and even genomic coverage." The researchers first needed to rule out that the DNA they had discovered was not ''Mycobacterium bovis, a related species. The samples were screened for the'' mtp40'' element, a common element in the phospholipase A gene of TB but not M. bovis. Additionally, the ''OxyR ''pseudogene was sequenced at position 285, where ''M. tuberculosis ''has a guanine residue and ''M. bovis ''has an adenine. Finally, the ''katG ''and ''gyrA ''genes were screened for SNP's unique to TB. These tests came back negative, confirming that the sample was in fact ''M. tuberculosis. '' The Illumina sequences taken from the mummy were compared to the H37Rv reference strain and the 7199/99 strain (from a TB outbreak in Germany 1998-2010). Spoligotyping (spacer oligonucleotide genotyping with PCR primers) revealed that there were two strains of TB within the mummy. Both mummified strains (M1 and M2 at right) contained 3 loci deletions and 300 SNP's compared to the H37Rv strain. M2 contained an additional 12 loci deletions and 398 SNP's from M1. The most "modern" of the strains, 7199/99, contained 3 more deletions and 164 more SNP's compared to M2. This data suggests that ''M. tuberculosis ''undergoes many deletions as it evolves. This study also supported the hypothesis that antibiotic resistant ''M. tuberculosis ''strains are a recent development, as these samples did not contain antibiotic resistance genes like those in the Beijing W strain. References Allen P. 2013. Metagenomic Analysis of Tuberculosis in a Mummy. ''N Engl J Med. ''3693, 289-290. Fletcher HA, Donoghue HD, Taylor GM, van der Zanden AGM, Spigelman M. 2003. "Molecular analysis of ''Mycobacterium tuberculosis ''DNA from a family of 18th century Hungarians." ''Microbiology. ''149, 143-151.